Method for the production of conjugates of insulin-like growth factor-1 and poly(ethylene glycol)

ABSTRACT

The present invention relates to a fusion protein comprising IGF-I or an IGF-I variant N-terminally linked to the C-terminus of a propeptide. The invention relates also to a method involving the use of the aforementioned fusion protein in the production of a lysine-PEGylated IGF-I or IGF-I variant. The method comprises the steps of cultivating a prokaryotic host cell comprising an expression vector containing a nucleic acid encoding the fusion protein and causing the cell to express the fusion protein, recovering and PEGylating said fusion protein, cleaving said PEGylated fusion protein with IgA protease, and recovering lysine-PEGylated IGF-I or IGF-I variant. The invention relates also to a lysine-PEGylated IGF-I or IGF-I variant produced using the above method. In addition, the invention relates to a method for treating a neurodegenerative disorders like Alzheimer&#39;s Disease using the lysine-PEGylated IGF-I or IGF-I variant and a composition comprising the lysine-PEGylated IGF-I or IGF-I variant.

PRIORITY TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/767,829, filed Apr. 27, 2010, now pending; which is a continuation ofU.S. application Ser. No. 12/576,266, filed Oct. 9, 2009, now abandoned;which is a division of U.S. application Ser. No. 11/846,857, filed Aug.29, 2007, now U.S. Pat. No. 7,625,996, issued Dec. 1, 2009; which claimsthe benefit of European Patent Application No. 06018170.8, filed Aug.31, 2006. The entire contents of the above-identified applications arehereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to a conjugate of insulin-like growth factor-I(IGF-I) or a variant thereof with poly(ethylene glycol) (PEG), a methodfor the production of such a conjugate, a composition comprising such aconjugate, a method for the use of such a conjugate, and a fusionprotein comprising IGF-I or an IGF-I variant N-terminally linked to theC-terminus of a propeptide which may be used in the method for makingthe above conjugate.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is an increasingly prevalent form ofneurodegeneration that accounts for approximately 50%-60% of the overallcases of dementia among people over 65 years of age. It currentlyaffects an estimated 15 million people worldwide and, owing to therelative increase of elderly people in the population, its prevalence islikely to increase over the next 2 to 3 decades. AD is a progressivedisorder with a mean duration of around 8.5 years between onset ofclinical symptoms and death. Death of pyramidal neurons and loss ofneuronal synapses in brain regions associated with higher mentalfunctions results in the typical symptoms, characterized by gross andprogressive impairment of cognitive function (Francis, P. T., et al., J.Neurol. Neurosurg. Psychiatry 66 (1999) 137-147). AD is the most commonform of both senile and presenile dementia in the world and isrecognized clinically as relentlessly progressive dementia that presentswith increasing loss of memory, intellectual function and disturbancesin speech (Merritt, A Textbook of Neurology, 6th ed., Lea& Febiger,Philadelphia (1979), pp. 484-489). Neuropathologically, the majorhallmarks of AD are the presence of two characteristic lesions: theamyloid senile plaque and neurofibrillary tangle (NFT). While the plaqueis deposited extraneuronally, the tangle is observed intraneuronally inthe post-mortem brain. One of the major components of the amyloid plaquecore is the pathologically deposited small amyloid-beta-peptide (Aβ),which is cleaved by secretases from amyloid precursor protein (APP)(Selkoe, D. J., Physiol. Rev. 81 (2001) 741-766; Hardy, J. and Selkoe,D. J., Science 297 (2002) 353-356; Bush, A. I. and Tanzi, R. E., Proc.Natl. Acad. Sci. USA 99 (2002) 7317-7319). Aβ (Abeta), aself-aggregating peptide of 39-43 residues (MW ˜4 kDa), is synthesizedas part of the larger APP (110-120 kDa). APP is a type I integralmembrane glycoprotein with a large N-terminal extracellular domain, asingle transmembrane domain and a short cytoplasmic tail. The Aβ regionspans portions of the extracellular and transmembrane domains of APP.The most common hypothesis for the participation of APP in neuronal celldeath in AD is the amyloid hypothesis. This hypothesis postulates thatplaque amyloid depositions or partially aggregated soluble Aβ trigger aneurotoxic cascade, thereby causing neurodegeneration similar to ADpathology (Selkoe, D. J., Physiol. Rev. 81 (2001) 741-766; Hardy, J. andSelkoe, D. J., Science 297 (2002) 353-356).

Human insulin-like growth factor I (IGF-I) is a circulating hormonestructurally related to insulin. IGF-I was traditionally considered themajor mediator of the actions of growth hormone on peripheral tissues.IGF-I consists of 70 amino acids and is also named somatomedin C anddefined by SwissProt No. P01343. Use, activity and production arementioned in, e.g., le Bouc, Y., et al., FEBS Lett. 196 (1986) 108-112;de Pagter-Holthuizen, P., et al., FEBS Lett. 195 (1986) 179-184;Sandberg Nordqvist, A. C., et al., Brain Res. Mol. Brain. Res. 12 (1992)275-277; Steenbergh, P. H., et al., Biochem. Biophys. Res. Commun. 175(1991) 507-514; Tanner, J. M., et al., Acta Endocrinol. (Copenh.) 84(1977) 681-696; Uthne, K., et al., J. Clin. Endocrinol. Metab. 39 (1974)548-554; EP 0 123 228; EP 0 128 733; U.S. Pat. No. 5,861,373; U.S. Pat.No. 5,714,460; EP 0 597 033; WO 02/32449; WO 93/02695.

The regulation of IGF-I function is quite complex. In the circulation,only 0.2% of IGF-I exists in the free form whereas the majority is boundto IGF-binding proteins (IGFBP's), which have very high affinities toIGF's and modulate IGF-I function. The factor can be locally liberatedby mechanisms releasing IGF-I such as proteolysis of IGFBPs byproteases.

IGF-I plays a paracrine role in the developing and mature brain(Werther, G. A., et al., Mol. Endocrinol. 4 (1990) 773-778). In vitrostudies indicate that IGF-I is a potent non-selective trophic agent forseveral types of neurons in the CNS (Knusel, B., et al., J. Neurosci. 10(1990) 558-570; Svrzic, D., and Schubert, D., Biochem. Biophys. Res.Commun. 172 (1990) 54-60), including dopaminergic neurons (Knusel, B.,et al., J. Neurosci. 10 (1990) 558-570) and oligodendrocytes (McMorris,F. A., and Dubois-Dalcq, M., J. Neurosci. Res. 21 (1988) 199-209;McMorris, F. A., et al., Proc. Natl. Acad. Sci. USA 83 (1986) 822-826;Mozell, R. L., and McMorris, F. A., J. Neurosci. Res. 30 (1991)382-390)). U.S. Pat. No. 5,093,317 mentions that the survival ofcholinergic neuronal cells is enhanced by administration of IGF-I. It isfurther known that IGF-I stimulate peripheral nerve regeneration (Kanje,M., et al., Brain Res. 486 (1989) 396-398) and enhance ornithinedecarboxylase activity U.S. Pat. No. 5,093,317). U.S. Pat. No. 5,861,373and WO 93/02695 mention a method of treating injuries to or diseases ofthe central nervous system that predominantly affects glia and/ornon-cholinergic neuronal cells by increasing the active concentration(s)of IGF-I and/or analogues thereof in the central nervous system of thepatient. WO 02/32449 is directed to methods for reducing or preventingischemic damage in the central nervous system of a mammal byadministering to the nasal cavity of the mammal a pharmaceuticalcomposition comprising a therapeutically effective amount of IGF-I orbiologically active variant thereof. The IGF-I or variant thereof isabsorbed through the nasal cavity and transported into the centralnervous system of the mammal in an amount effective to reduce or preventischemic damage associated with an ischemic event. EP 0 874 641 claimsthe use of an IGF-I or an IGF-II for the manufacture of a medicament fortreating or preventing neuronal damage in the central nervous system,due to AIDS-related dementia, AD, Parkinson's Disease, Pick's Disease,Huntington's Disease, hepatic encephalopathy, cortical-basal ganglionicsyndromes, progressive dementia, familial dementia with spasticparapavresis, progressive supranuclear palsy, multiple sclerosis,cerebral sclerosis of Schilder or acute necrotizing hemorrhagicencephalomyelitis, wherein the medicament is in a form for parenteraladministration of an effective amount of said IGF outside theblood-brain barrier or blood-spinal cord barrier.

Reduction of brain and serum levels of free IGF-I has been related tothe pathogenesis of sporadic and familial forms of AD. Furthermore,IGF-I protects neurons against Aβ-induced neurotoxicity (Niikura, T., etal., J. Neurosci. 21 (2001) 1902-1910; Dore, S., et al., Proc. Natl.Acad. Sci. USA 94 (1997) 4772-4777; Dore, S., et al., Ann. NY Acad. Sci.890 (1999) 356-364). Recently, it was shown that peripherallyadministered IGF-I is capable of reducing brain Aβ levels in rats andmice (Carro, E., et al., Nat. Med. 8 (2002) 1390-1397). Furthermore, thestudy demonstrated that in a transgenic AD mouse model prolonged IGF-Itreatment significantly reduced brain amyloid plaque load. These datastrongly support the idea that IGF-I is able to reduce brain Aβ levelsand plaque-associated brain dementia by clearing Aβ from the brain.

Covalent modification of proteins with poly(ethylene glycol) (PEG) hasproven to be a useful method to extend the circulating half-lives ofproteins in the body (Hershfield, M. S., et al., N. Engl. J. Med. 316(1987) 589-596; Meyers, F. J., et al., Clin. Pharmacol. Ther. 49 (1991)307-313; Delgado, C., et al., Crit. Rev. Ther. Drug Carrier Syst. 9(1992) 249-304; Katre, Advanced Drug Delivery Reviews 10 (1993) 91-114;EP-A 0 400 472; Monfardini, C., et al., Bioconjugate Chem. 6 (1995)62-69; Satake-Ishikawa, R., et al., Cell Struct. Funct. 17 (1992)157-160; Katre, N. V., et al., Proc. Natl. Acad. Sci. USA 84 (1987)1487-1491; Tsutsumi, Y., et al., Jpn. J. Cancer Res. 85 (1994) 9-12;Inoue, H., et al., J. Lab. Clin. Med. 124 (1994) 529-536; Chamow, S. M.,et al., Bioconjugate Chem. 5 (1994) 133-140).

Other advantages of PEGylation are an increase of solubility and adecrease in protein immunogenicity (Katre, N. V., J. Immunol. 144 (1990)209-213). A method for the PEGylation of proteins is the use ofpoly(ethylene glycol) activated with amino-reactive reagents likeN-hydroxysuccinimide (NHS). With such reagents poly(ethylene glycol) isattached to the proteins at free primary amino groups such as theN-terminal α-amino group and the E-amino groups of lysine residues. Alimitation of this approach is that proteins typically contain aconsiderable amount of lysine residues and therefore the poly(ethyleneglycol) groups are attached to the protein in a non-specific manner atall of the free E-amino groups, resulting in a heterologous productmixture of random PEGylated proteins. Therefore, NHS-PEGylated proteinscan be unsuitable for commercial use because of low specific activity.Inactivation results from covalent modification of one or more lysineresidues or the N-terminal amino residue required for biologicalactivity or from covalent attachment of the poly(ethylene glycol)residues near or at the active site of the protein. For example, it wasfound that modification of human growth hormone using NHS-PEGylationreagents reduces the biological activity of the protein by more than10-fold (Clark, R., et al., J. Biol. Chem. 271 (1996) 21969-21977).Human growth hormone contains 9 lysines in addition to the N-terminalamino acid. Certain of these lysines are located in regions of theprotein known to be critical for receptor binding (Cunningham, B. C., etal., Science 254 (1991) 821-825). In addition, the modification oferythropoietin by the use of amino-reactive poly(ethylene glycol)reagents results also in a nearly complete loss of biological activity(Wojchowski, D. M., et al., Biochim. Biophys. Acta 910 (1987) 224-232).Covalent modification of Interferon-α2 with amino-reactive PEGylationreagents results in 40-75% loss of bioactivity (U.S. Pat. No.5,382,657). A similar modification of G-CSF results in greater than 60%loss of activity (Tanaka, H., et al., Cancer Res. 51 (1991) 3710-3714)and of Interleukin-2 in greater than 90% loss of bioactivity (Goodson,R. J., and Katre, N. V., BioTechnology 8 (1990) 343-346).

WO 94/12219 and WO 95/32003 claim polyethylene glycol conjugatescomprising PEG and IGF or a cystein mutated IGF, said PEG attached tosaid mutein at a free cystein in the N-terminal region of the mutein. WO2004/60300 describes N-terminally PEGylated IGF-I.

The recognition site of the IgA Protease is described asYaa-Pro.!.Xaa-Pro (as used herein, “.!.” refers to the cleavage site forIgA Protease). Yaa stands for Pro (or rarely for Pro in combination withAla, Gly or Thr: Pro-Ala, Pro-Gly, or Pro-Thr. Xaa stands for Thr, Seror Ala (Pohlner, J., et al., Bio/Technology 10 (1992) 799-804; Pohlner,J., et al., Nature 325 (1987) 458-462; and U.S. Pat. No. 5,427,927).Natural cleavage sites have been identified by Wood, S. G. and BurtonJ., Infect. Immun. 59 (1991) 1818-1822. Synthetic peptide substrates forthe immunoglobulin A1 protease from Neisseria gonorrhoea (type 2) arethe autoproteolytic sites Lys-Pro-Ala-Pro.!.Ser-Pro (SEQ ID NO: 30),Val-Ala-Pro-Pro.!.Ser-Pro (SEQ ID NO: 31), Pro-Arg-Pro-Pro.!.Ala-Pro(SEQ ID NO: 32), Pro-Arg-Pro-Pro.!.Ser-Pro (SEQ ID NO: 33),Pro-Arg-Pro-Pro.!.Thr-Pro (SEQ ID NO: 34) and the IgA1 Cleavage SitesPro-Pro-Thr-Pro.!.Ser-Pro (SEQ ID NO: 35) and Ser-Thr-Pro-Pro.!.Thr-Pro(SEQ ID NO: 36).

WO 2006/066891 discloses conjugates consisting of an insulin-like growthfactor-1 (IGF-I) variant and one or two poly(ethylene glycol) group(s),characterized in that said IGF-I variant has an amino acid alteration atup to three amino acid positions 27, 37, 65, 68 of the wild-type IGF-Iamino acid sequence so that one or two of said amino acids is/are lysineand amino acid 27 is a polar amino acid but not lysine, is conjugatedvia the primary amino group(s) of said lysine(s) and said poly(ethyleneglycol) group(s) have an overall molecular weight of from 20 to 100 kDais disclosed. Such conjugates are useful for the treatment ofneurodegenerative disorders like Alzheimer's Disease. WO 2006/074390refers to IGF-I fusion polypeptides.

SUMMARY OF THE INVENTION

The present invention relates in part to a fusion protein whichcomprises IGF-I or an IGF-I variant N-terminally linked to theC-terminus of a propeptide. The C-terminus of the propeptide comprisesthe amino acid sequence Y-Pro, wherein Y is selected from the groupconsisting of: Pro-; Pro-Ala; Pro-Gly; Pro-Thr; Ala-Pro; Gly-Pro;Thr-Pro; Arg-Pro; and Pro-Arg-Pro. The amino acid residue(s) of “Y”,above, constitute(s) a fragment of a lysine-free beta-galactosidase. Thecorresponding nucleic acid sequence encoding this fragment improves theexpression of the fusion protein.

Another aspect of the present invention is a method for the productionof a lysine-PEGylated IGF-I or a lysine-PEGylated IGF-I variant whichcomprises the steps of:

-   a) cultivating a prokaryotic host cell comprising an expression    vector containing a nucleic acid encoding the aforementioned fusion    protein and causing said cell to express said fusion protein,-   b) recovering and PEGylating said fusion protein,-   c) cleaving said PEGylated fusion protein with IgA protease, and-   d) recovering said lysine-PEGylated IGF-I or lysine-PEGylated IGF-I    variant.

A further aspect of the present invention is a lysine-PEGylated IGF-I orlysine-PEGylated IGF-I variant produced using the above method.

A further aspect of the present invention is a composition whichcomprises a lysine-PEGylated IGF-I or lysine-PEGylated IGF-I variantaccording to the invention, preferably together with a pharmaceuticallyacceptable carrier.

Yet another aspect of the present invention is a method for theproduction of the aforementioned composition.

The invention further relates to a method for the treatment ofAlzheimer's disease comprising the administration of apharmaceutically-effective amount of a lysine-PEGylated IGF-I (SEQ IDNO: 1) or a lysine-PEGylated IGF-I variant to a patient in need of suchtreatment.

DESCRIPTION OF THE SEQUENCE LISTING

-   -   SEQ ID NO: 1 amino acid sequence of human IGF-I (amino acids        49-118 from SwissProt P01343).    -   SEQ ID NO: 2 amino acid sequence of fusion protein px3036_IAG_R        K27R K65R K68    -   SEQ ID NO: 3 amino acid sequence of fusion protein        px3036_IAEE_F1 K27R K65R K68    -   SEQ ID NO: 4 amino acid sequence of fusion protein        px3036_IAFX_F1 K27R K65R K68    -   SEQ ID NO: 5 amino acid sequence of fusion protein px3036        JAFX_F2 K27R K65R K68    -   SEQ ID NOs: 6-10 linker

SEQ ID NOs: 11-18 cleavage sequences

SEQ ID NOs: 19-21 sequences for use as X₁-His_(n)

-   -   SEQ ID NO: 22 amino acid sequence of fusion protein px3036_IAG_R        K27R K65 K68R    -   SEQ ID NO: 23 amino acid sequence of fusion protein        px3036_IAEE_F1 K27R K65 K68R    -   SEQ ID NO: 24 amino acid sequence of fusion protein        px3036_IAFX_F1 K27R K65        -   K68R    -   SEQ ID NO: 25 amino acid sequence of fusion protein        px3036_IAFX_F2 K27R K65        -   K68R    -   SEQ ID NO: 26 amino acid sequence of the IGF-I variant RKK    -   SEQ ID NO: 27 amino acid sequence of the IGF-I variant RKR    -   SEQ ID NO: 28 amino acid sequence of the IGF-I variant RRK    -   SEQ ID NO: 29 amino acid sequence of a fragment of a lusine-free        beta-galactosidase    -   SEQ ID NOs: 30-36 amino acid sequences of synthetic peptide        substrates for the immunoglobulin A1 protease from Neisseria        gonorrhoea.

DESCRIPTION OF THE FIGURES

FIG. 1: Peptide Analysis of monoPEGylated Fusion Protein.

SDS-PAGE analysis of folded fusion protein before and after PEGylation.Lane 1, mixture of standard protein (bovine lung aprotinin, 6.0 kDa;chicken egg white lysozyme, 14.4 kDa; soybean trypsin inhibitor, 21.5kDa; bovine erythrocyte carbonic anhydrase, 31.0 kDa; porcine musclelactate dehydrogenase, 36.5 kDa; bovine liver glutamic dehydrogenase,55.4 kDa; bovine serum albumin, 66.3 kDa; rabbit muscle phosphorylase b,97.4 kDa; E. coli β-galactosidase, 97.4 kDa; rabbit muscle myosin, 200kDa); lane 2, pro-IGF-I before pegylation; lane 3, pro-IGF-I afterpegylation.

FIG. 2: Peptide Analysis of monoPEGylated IGF-I Variant.

SDS-PAGE analysis of pegylated fusion protein before and after IgAcleavage. Lane 1, mixture of standard protein (same as in FIG. 1); lane2, reaction mix before IgA protease cleavage; lane 3, reaction mix afterIgA protease cleavage.

FIG. 3: SDS PAGE.

SDS-PAGE analysis of folded fusion protein before and after PEGylation.

Lane 1, mixture of standard protein (same as in FIG. 1); lane 2,reaction mix before IEC; lane 3, flow through of IEC; lanes 4-19, singleeluted fractions from IEC.

FIG. 4: In Vivo Brain Abeta Lowering of PEG-RRK in B6.152H Mice.

Double-transgenic B6.152H mice aged 9-10 months were treated withvehicle (NaCl) or with PEG-RRK (5 μg/kg s.c., twice-a-week) for 14 days.Soluble brain extracts were prepared and APP, Abeta and Actin levelsevaluated as described. The ratios APP/Actin, Abeta/Actin and Abeta/APPwere calculated, are expressed as % of control. A, APP/Actin; B,Abeta/Actin, C, Abeta/APP. Upper graphs show single animal data points,lower graphs show bar representation (means±SEM) including statisticaldifferences (*, p<0.05; **, p<0.01 vs. untreated control, n=10).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “IGF-I” refers to human insulin-like growth factor. Itconsists of 70 amino acids and has the amino acid sequence of SEQ ID NO:1.

The present invention relates in part to a fusion protein whichcomprises IGF-I (SEQ ID NO: 1) or an IGF-I variant N-terminally linkedto the C-terminus of a propeptide. The C-terminus of the propeptidecomprises the amino acid sequence Y-Pro, wherein Y is selected from thegroup consisting of: Pro; Pro-Ala; Pro-Gly; Pro-Thr; Ala-Pro; Gly-Pro;Thr-Pro; Arg-Pro; Pro-Arg-Pro; Ala-Pro-Arg-Pro (SEQ ID NO: 12); andPro-Ala-Pro-Arg-Pro (SEQ ID NO: 13). In preferred embodiments, Y isselected from the group consisting of: Pro; Pro-Ala; Arg-Pro;Pro-Arg-Pro Ala-Pro-Arg-Pro (SEQ ID NO: 12); and Pro-Ala-Pro-Arg-Pro(SEQ ID NO: 13). In especially preferred embodiments, Y is selected fromthe group consisting of: Pro; Pro-Arg-Pro; Ala-Pro-Arg-Pro (SEQ ID NO:12); and Pro-Ala-Pro-Arg-Pro (SEQ ID NO: 13).

The IGF-I variant is a polypeptide consisting of 70 amino acids thatdiffers from IGF-I (SEQ ID NO: 1) in that one or two of the lysinespresent at residue positions 27, 65, and 68 of IGF-I is independentlysubstituted by a polar amino acid selected from the group consisting of:cysteine (C); aspartic acid (D); glutamic acid (E); histidine (H);asparagine (N); glutamine (Q); arginine (R); serine (S); and threonine(T). In preferred embodiments, the aforementioned lysine(s) is/aresubstituted with arginine(s). In each case of an IGF-I variant, theremainder of the amino acid sequence of the IGF-I variant is the same asthat of IGF-I. In preferred embodiments, the polar amino acid isselected from the group consisting of: arginine; glutamine; andasparagine. In especially preferred embodiments, the polar amino acid isasparagine.

In preferred embodiments, the IGF-I variant is selected from the groupconsisting of: RRK, RKR, and RRK. As used herein, “RKK” refers to anIGF-I variant of SEQ ID NO: 26 and differs from IGF-I (SEQ ID NO: 1) inthat it contains arginine instead of lysine at residue position 27,“RKR” refers to an IGF-I variant of SEQ ID NO: 27 and differs from IGF-I(SEQ ID NO: 1) in that it contains arginine instead of lysine at residuepositions 27 and 68, and “RRK” refers to an IGF-I variant of SEQ ID NO:28 and differs from IGF-I (SEQ ID NO: 1) in that it contains arginineinstead of lysine at residue positions 27 and 65. In IGF-I, lysine ispresent at residue positions 27, 65, and 68 and, therefore, “KKK” may beused to refer to IGF-I.

In preferred embodiments, the propeptide does not comprise a lysineresidue.

In a preferred embodiment, the fusion protein comprises up to 30 aminoacids.

In a preferred embodiment, the fusion protein has the following aminoacid sequenceMet-X₁-His_(n)-X₂-Pro-Z

wherein:

-   -   Met is a methionine residue;    -   X₁ is selected from the group consisting of: a bond, serine, or        asparagine;    -   His is a histidine residue;    -   n is an integer from 0 to 10;    -   X₂ is a linker peptide having an amino acid sequence selected        from the group consisting of: SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID        NO: 8; SEQ ID NO: 9; and SEQ ID NO: 10;    -   Pro is a proline residue; and    -   Z is IGF-I (SEQ ID NO: 1) or an IGF-I variant as described        above.

The present invention relates also to a method for the production of alysine-PEGylated IGF-I or a lysine-PEGylated IGF-I variant. As usedherein, the term “PEGylation” refers to the attachment of a PEG group orPEG groups to a molecule and a molecule is said to be “PEGylated” if ithas a PEG group or PEG groups attached. “Lysine-PEGylated”, when used torefer to IGF-I (SEQ ID NO: 1) or an IGF-I variant, means that the IGF-I(SEQ ID NO: 1) or IGF-I variant has a PEG group attached to a lysineresidue therein. The IGF-I variant is as described above.

As used herein, “PEG” refers to poly(ethylene glycol). As used herein,the terms “poly(ethylene) glycol” and “PEG” broadly encompass anypoly(ethylene glycol) molecule in which the number of ethylene glycolunits is at least 460, preferably 460 to 2300 (about 20 kDa to 100 kDa)and especially preferably 460 to 1840. Poly(ethylene glycol) is awater-soluble polymer that is commercially available or can be preparedby ring-opening polymerization of ethylene glycol according to methodswell known in the art (Kodera, Y., et al., Progress in Polymer Science23 (1998) 1233-1271; Francis, G. E., et al., Int. J. Hematol. 68 (1998)1-18). The number of ethylene glycol units in PEG is approximated forthe molecular mass described in Daltons. For example, if two PEGmolecules are attached to a linker where each PEG molecule has the samemolecular mass of 10 kDa, then the total molecular mass of PEG on thelinker is about 20 kDa. The molecular masses of the PEG attached to thelinker can also be different, e.g., of two molecules on a linker one PEGmolecule can be 5 kDa and one PEG molecule can be 15 kDa.

Usually PEGs which contain more than 2300 ethylene glycol units are notused. Preferably, a PEG used in the invention terminates on one end withhydroxy or methoxy (methoxy PEG, mPEG) and is, on the other end,covalently attached to a linker moiety via an ether oxygen bond. The PEGpolymer is either linear or branched. Preferably the PEG polymer isbranched. Branched PEGs are described, e.g., in Veronese, F. M., et al.,Journal of Bioactive and Compatible Polymers 12 (1997) 196-207. UsefulPEG reagents are, e.g., available from Nektar Therapeutics(www.nektar.com). Branched PEGs can be prepared, for example, by theaddition of polyethylene oxide to various polyols, including glycerol,pentaerythriol, and sorbitol. For example, a four-armed branched PEG canbe prepared from pentaerythriol and ethylene oxide. Branched PEGsusually have 2 to 8 arms and are described in, for example, EP-A 0 473084 and U.S. Pat. No. 5,932,462. Especially preferred are PEGs with twoPEG side-chains linked via the primary amino group of a lysine(abbreviated as PEG2) (Monfardini, C., et al., Bioconjugate Chem. 6(1995) 62-69).

It was surprisingly found that IgA protease is capable of cleaving theamino acid sequence Y-Pro.!.Gly-Pro (“.!.” herein and hereafterrepresents the cleavage position) wherein Y is as described above andGly-Pro are the first two amino acids of IGF-I (SEQ ID NO: 1) or theIGF-I variant. As discussed above, in preferred embodiments Y isselected from the group consisting of: Pro; Pro-Ala; Pro-Gly; Pro-Thr;Ala-Pro; Gly-Pro; Thr-Pro; Arg-Pro; Pro-Arg-Pro; Ala-Pro-Arg-Pro (SEQ IDNO: 12); and Pro-Ala-Pro-Arg-Pro (SEQ ID NO: 13). Accordingly, therespective sequences around the cleavage site include:Pro-Ala-Pro.!.Gly-Pro (SEQ ID NO:14), Pro-Pro-!.Gly-Pro (SEQ ID NO:15),Pro-Arg-Pro-Pro.!. Gly-Pro (SEQ ID NO:16), Ala-Pro-Arg-Pro-Pro.!.Gly-Pro(SEQ ID NO:17) and Pro-Ala-Pro-Arg-Pro-Pro.!.Gly-Pro (SEQ ID NO:18).

In accordance with the present invention the term “IgA protease”includes proteases which specifically cleave IgA and which are describedfor example by Kornfeld, S. J. and Plaut, A. G. in Rev. Infekt. Dis. 3(1981) 521-534 as e.g. IgA1 protease from Neisseria gonorrhoea (type 2).Recombinant IgA proteases such as those described in DE-A 36 22 221;Koomey, J. M., et al., Proc. Natl. Acad. Sci. USA 79 (1982) 7881-7885;Bricker, J., et al., Proc. Natl. Acad. Sci. USA 80 (1983) 2681-2685;Pohlner, J., et al., Nature 325 (1987) 458-462; and Halter, R., et al.,EMBO J. 3 (1984) 1595-1601 are also just as suitable. Preferably saidIgA protease is IgA protease from Neisseria gonorrhoea, preferably type2.

The method of the present invention therefore relates to a methodcomprising the steps of expressing the aforementioned fusion protein,recovering the fusion protein, and cleaving the fusion protein using IgAprotease to recover lysine-PEGylated IGF-I or lysine-PEGylated IGF-Ivariant.

The gene encoding the fusion protein is preferably placed under thecontrol of suitable (preferably inducible) expression signals so thatfusion proteins can be produced according to the requirements. Suitableprokaryotic or eukaryotic (plant as well as animal) cells can be used ashost cells for the production of protein fusions; cell-free systems are,however, also possible.

A preferred embodiment of the process according to the present inventionis characterized in that a host cell is transformed with a recombinantDNA or a recombinant vector, in which the DNA or the vector contains atleast one copy of a gene which codes for a fusion protein according tothe invention and the transformed cell is cultured in a suitable medium,the gene coding for the fusion protein is made to express in thetransformed cell, the fusion protein is PEGylated and subsequentlycleaved with IgA protease and the PEGylated IGF-I or IGF-I variant isisolated.

The method of the present invention for the production of alysine-PEGylated IGF-I or a lysine PEGylated IGF-I variant thereforecomprises the steps of:

-   -   (A) cultivating a prokaryotic host cell comprising an expression        vector containing a nucleic acid encoding a fusion protein        according to claim 1 and causing said cell to express said        fusion protein;    -   (B) recovering and PEGylating said fusion protein;    -   (C) cleaving said PEGylated fusion protein with IgA protease,        and    -   (D) recovering said lysine-PEGylated IGF-I or lysine-PEGylated        IGF-I variant.

It is known in the art that the production of recombinant protein inbacteria, such as E. coli., may lead to the addition of a methionineresidue at the N-terminus which may alter the biological activity of theprotein. Moriyama et al., Comp. Biochem. Physiol., 117: 201-206 (1997).An advantage to using the method of the present invention is that theresulting lysine-PEGylated IGF-I or lysine-PEGylated IGF-I variant doesnot have an N-terminal methionine.

The expression of the fusion protein according to the invention can, forexample, be improved at the DNA level by the inclusion of fragments oflysine-free beta-galactosidase gene, i.e., Y (as defined previously)contains a part of a lysine-free beta-galactosidase protein. Otheralternatives for increasing the expression of the fusion protein areknown to the expert. The purification and separation of the expressionproduct can be facilitated by the inclusion in the fusion protein ofcertain polypeptides, in particular, polypeptides or proteins that arehighly charged (e.g. poly(Lys, Arg)) or which can bind to particularsubstances with high affinity (e.g. streptavidin) (see e.g. EP-A 0 089626, EP-A 0 306 610). Especially preferred are linker peptides of SEQ IDNO: 6; SEQ ID NO: 7; SEQ ID NO: 8: SEQ ID NO: 9; and SEQ ID NO: 10;preferably N-terminally preceded by SHHHHHH (SEQ ID NO:19), NHHHHHH (SEQID NO:20) or HHHHHH (SEQ ID NO:21) (S=serine; N=asparagine; H=histidine)

The present invention also provides a (recombinant) nucleic acid whichcodes for a fusion protein according to the present invention in whichan IgA protease cleavage site is incorporated in the junction regionbetween the propeptide and the IGF-I or IGF-I variant.

A recombinant DNA according to the present invention can be obtained ina manner known to one skilled in the art of molecular biology. For this,a vector which contains a DNA sequence coding for the amino acidsequence of the IGF-I or IGF-I variant is usually cleaved withrestriction endonuclease(s) in the region of the 5′ end of this gene andreligated with oligonucleotides which contain the desired sequence.

In addition, the invention also provides a recombinant vector whichcontains at least one copy of a recombinant DNA according to the presentinvention. Vectors which are suitable as a basis for protein expressionin prokaryotic organisms are known to the expert. This vector ispreferably one which allows a high expression of the recombinant DNAaccording to the present invention. The recombinant DNA on the vector ispreferably under the control of an inducible expression signal (e.g.lambda., tac, lac or trp promoter).

The vector according to the present invention can be presentextrachromosomally (e.g. plasmid) or can be integrated in the genome ofthe host organism (e.g. bacteriophage lambda). The vector according tothe present invention is preferably a plasmid. Vectors which aresuitable in each case for gene expression in a particular host organismare known to one skilled in the art of molecular biology. It can be aeukaryotic vector, but preferably is a prokaryotic vector. Examples ofsuitable vectors for the expression of the DNA according to the presentinvention in prokaryotes are, for instance, commercially available pUCand pUR vectors.

The invention also provides a cell, preferably a prokaryotic cell,particularly preferably an E. coli cell, which is transformed with therecombinant DNA according to the present invention or/and with arecombinant vector according to the present invention.

When the fusion protein is expressed in prokaryotes, sparingly solubleaggregates (refractile bodies, inclusion bodies) are formed which areinactive. Therefore the fusion protein must be transformed into itsactive form. Using procedures which are familiar to those skilled in theart (cf. e.g. EP-A 0 219 874, EP A 0 114 506, WO 84/03711), first asolubilization is carried out by addition of denaturing agents which isfollowed by renaturation and, if desired, further purification steps.The treatment of the fusion protein with IgA protease takes place afterPEGylation of the fusion protein.

In PEGylation, PEG groups are attached to reactive primary ε-aminogroups of lysine residues of the fusion protein and optionally theα-amino group of the N-terminal amino acid of the fusion protein. Suchamino group attachment of PEG to proteins is well known in the art. Forexample, review of such methods is given by Veronese, F. M.,Biomaterials 22 (2001) 405-417. According to Veronese, the attachment ofPEG to primary amino groups of proteins can be performed by usingactivated PEG derivatives which perform an alkylation of said primaryamino groups.

Activated PEG derivatives are known in the art and are described in, forexample, Morpurgo, M., et al., J. Bioconjugate Chem. 7 (1996) 363-368for PEG-vinylsulfone. Examples of such derivatives includeiodo-acetyl-methoxy-PEG and methoxy-PEG-vinylsulfone. The use of theseiodo-activated substances is known in the art and is described, e.g., byHermanson, G. T., in Bioconjugate Techniques, Academic Press, San Diego(1996), pp. 147-148. Electrophilically-activated PEG derivatives such asalkoxybutyric acid succinimidyl esters of poly(ethylene glycol) (“loweralkoxy-PEG-SBA”), alkoxypropionic acid succinimidyl esters ofpoly(ethylene glycol) (“lower alkoxy-PEG-SPA”), and N-hydroxysuccinimideactivated PEGs are preferred. Further examples of activated PEGderivatives which may be used in PEGylation include: alkylating PEGderivatives, for example PEG aldehyde, PEG-tresyl chloride or PEGepoxide; acylating PEG derivatives such as hydroxysuccinimidyl esters ofcarboxylated PEGs or PEGs in which the terminal hydroxy group isactivated by chloroformates or carbonylimidazole; and PEGs with aminoacid arms. Such derivatives can contain the so-called branched PEGs,whereby at least two identical or different PEG molecules are linkedtogether by a peptidic spacer (preferably lysine) and, for example,bound to IGF-I or IGF-I variant as activated carboxylate of the lysinespacer. N-hydroxysuccinimidyl esters of PEG and, preferably,N-hydroxysuccinimidyl esters of methoxypoly(ethylene glycol) arepreferred activated PEG derivatives. The use of succinimidyl esters toproduce conjugates with proteins is disclosed in U.S. Pat. No.5,672,662. An especially preferred activated PEG derivative for use inthe present invention is an N-hydroxysuccinimidyl activated branched PEGester (mPEG2-NHS).

In the examples below, some preferred reagents for the production ofamino-reactive IGF-I or IGF-I variants are described. It is understoodthat modifications, for example, based on the methods described byVeronese, F. M., Biomaterials 22 (2001) 405-417, can be made in theprocedures as long as the process results in lysine-PEGylated IGF-I orIGF-I variants according to the invention.

Lysine-PEGylated IGF-I or IGF-I variants according to the invention maybe prepared by covalently reacting a primary lysine amino group of anIGF-I or IGF-I variant with a bifunctional reagent to form anintermediate with an amide linkage and covalently reacting theintermediate containing amide linkage with an activated poly(ethyleneglycol) derivative to form a lysine-PEGylated IGF-I or IGF-I variant. Inthe foregoing process, the bifunctional reagent is preferablyN-succinimidyl-5-acetylthiopropionate orN-succinimidyl-5-acetylthioacetate, and the activated poly(ethyleneglycol) derivative is preferably selected from the group consisting ofiodo-acetyl-methoxy-PEG, methoxy-PEG-vinylsulfone, andmethoxy-PEG-maleimide.

Any conventional method of reacting an activated ester with an amine toform an amide can be utilized. In the reaction of anN-hydroxysuccinimidyl ester of PEG with IGF-I or an IGF-I variant, theexemplified succinimidyl ester is a leaving group causing the formationof an amide bond between IGF-I or IGF-I variant and PEG.

The reaction conditions used in PEGylation have an influence on therelative amount of differently PEGylated IGF-I or IGF-I variants. Bymanipulating the reaction conditions (e.g., ratio of reagents, pH,temperature, protein concentration, time of reaction etc.), the relativeamounts of the different PEGylated species can be varied. Preferably thereaction is performed in a buffered aqueous solution pH 8-10, optionallycontaining up to 30% (v/v) ethanol. The molar protein:PEG ratio ispreferably 1:1 to 1:6, preferably 1:2 to 1:5. Reaction temperature andreaction time can be varied according to the knowledge of a skilledartisan, whereby high temperature and long reaction time results inincreased PEGylation. If monoPEGylated proteins are desired, it ispreferred to work between 4° C. and 22° C. and for up to 30 minutes orup to 60 minutes. When PEG or an activated PEG derivative is combinedwith IGF-I or IGF-I variant in a reaction buffer which preferablyconsists of 50 mM sodium borate and 25% ethanol at a pH of about9.0-9.5, a protein:PEG ratio of about 1:3 to 1:4, and a reactiontemperature of 4° C., a mixture of mono-, di-, and trace amounts of thetri-PEGylated species is produced depending on the presence of lysineresidues in the protein.

The conditions required for the treatment of PEGylated IGF-I or IGF-Ivariant to be cleaved with IgA proteases are not critical. In thisprocess it is, however, preferred that the ratio by weight of PEGylatedIGF-I or IGF-I variant to IgA protease is 1:1 to 500:1, preferably,100:1. The reaction preferably takes place in a buffered aqueoussolution of pH 6.5 to 8.5. The buffer concentration is preferably in therange between 50 and 500 mmol/l if desired, with addition of 0-100mmol/l sodium chloride. The cleavage is preferably carried out at roomtemperature for at least 60 min up to 5 days, preferably between 24 and72 hours.

After solubilization, renaturation, PEGylation and cleavage with IgAprotease, the PEGylated cleavage product obtained in this way ispreferably purified by means of ion exchange chromatography, hydrophobicinteraction chromatography and/or fractionation by size. The PEGylatedIGF-I or IGF-I variant produced in this way is free of methionine at theN-terminus, preferably free of other proteins, like non-PEGylated IGF-Ior IGF-I variant, and preferably free of N-terminal PEGylatedpropeptide, by 5% (w/w) or lower.

In preferred embodiments of the present invention, the IGF-I or IGF-Ivariant is mono-PEGylated or di-PEGylated. As used herein, the term“mono-PEGylated” means that the IGF-I or IGF-I variant has one PEG groupattached thereto and the term “di-PEGylated” means that the IGF-I orIGF-I variant has two PEG groups attached thereto.

The invention also provides lysine-PEGylated IGF-I and lysine-PEGylatedIGF-I variants as produced using the aforementioned method. Suchlysine-PEGylated IGF-I or IGF-I variants contain linear or branched PEGrandomly attached thereto, whereby the overall molecular weight of allPEG groups in the lysine-PEGylated IGF-I or IGF-I variant is preferablyabout 20 to about 80 kDa. It is obvious to a person skilled in the artthat small deviations from this range of molecular weight are possibleas long as the PEGylated IGF-I or IGF-I variant does show activity inlowering Abeta peptide levels in the brain. Also IGF-I and IGF-Ivariants wherein the overall molecular weight of all attached PEG groupsis more than 80 kDa have higher bioavailability. However, it is expectedthat such activity decreases as the molecular weight increases due toreduced IGF-I receptor activation and blood-brain barrier transport.Therefore, the range of 20 to 100 kDa for the overall molecular weightof all attached PEG groups has to be understood as the optimized rangefor a lysine-PEGylated IGF-I or IGF-I variant useful for an efficienttreatment of a patient suffering from Alzheimer's disease. As statedabove, such a lysine-PEGylated IGF-I or lysine-PEGylated IGF-I variantdoes not contain an N-terminal methionine.

Preferably produced is a monoPEGylated IGF-I variant, selected from thegroup consisting of RKK (SEQ ID NO: 26), RKR (SEQ ID NO: 27) and RRK(SEQ ID NO: 28) wherein the branched PEG group has a molecular weight of30-45, preferably 40-45 kDa (about 920 ethylene glycol units). Forexample, based on an average molecular weight of 44 kDa for PEG and amolecular weight of 7.6 kDa for IGF-I, the calculated average molecularweight for such a monoPEGylated IGF-I is about 51.6 kDa. Especiallypreferred is the use of an N-hydroxysuccinimidyl activated branched PEGester (mPEG2-NHS) of a molecular weight of 40 kDa (Monfardini, C., etal., Bioconjugate Chem. 6 (1995) 62-69; Veronese, F. M., et al., J.Bioactive Compatible Polymers 12 (1997) 197-207; U.S. Pat. No.5,932,462). Also preferably produced is a monoPEGylated IGF-I whereinthe PEG has an average molecular weight of 30 or 40 kDa.

The following PEGylated forms of IGF-1 or IGF-I variants are preferredproducts and available by the methods according to the invention:

-   -   a monoPEGylated IGF-I variant, preferably RRK (SEQ ID NO: 28) or        RKK (SEQ ID NO: 26), more preferably RRK (SEQ ID NO: 28),        wherein the PEG group has a molecular weight of 20 to 80 kDa        (460 to 1840 ethylene glycol units) and is attached to the        lysine at residue position 68;    -   a monoPEGylated IGF-I variant, preferably RKR (SEQ ID NO: 27) or        RKK (SEQ ID NO: 26), more preferably RKR (SEQ ID NO: 27),        wherein the PEG group has a molecular weight of 20 to 80 kDa        (460 to 1840 ethylene glycol units) and is attached to the        lysine at residue position 65;    -   a diPEGylated IGF-I variant, preferably RKK (SEQ ID NO: 26),        wherein the PEG groups have a molecular weight of about 10-50        kDa (230 to 1150 ethylene glycol units) each and are attached to        the lysines at residue positions 65 and 68;    -   a monoPEGylated IGF-I variant, preferably RRK (SEQ ID NO: 28) or        RKK (SEQ ID NO: 26), more preferably RRK (SEQ ID NO: 28),        comprising a PEG2 group having a molecular weight of 40 kDa        attached to the lysine at residue position 68;    -   a monoPEGylated IGF-I variant, preferably RKR (SEQ ID NO: 27) or        RKK (SEQ ID NO: 26), more preferably RKR (SEQ ID NO: 27),        comprising a PEG2 group having a molecular weight of 40 kDa        attached to the lysine at residue position 65;    -   a monoPEGylated IGF-I, wherein the PEG group has a molecular        weight of 20 to 80 kDa (460 to 1840 ethylene glycol units);    -   a diPEGylated IGF-I, wherein the PEG groups have a molecular        weight of about 10-50 kDa (230 to 1150 ethylene glycol units)        each; and    -   a monoPEGylated IGF-I, comprising a PEG2 group having a        molecular weight of 40 kDa.

Preparations of PEGylated IGF-I or IGF-I variants are substantiallyhomogeneous. The preparation may contain small amounts of unreacted(i.e., lacking PEG group) protein. As ascertained by peptide mapping,purity of the variant is at least 90% (w/w). Further purification ofsuch preparations, including the separation of mono- and/or diPEGylatedIGF-I or IGF-I variants, can be performed by usual purification methods,preferably by size exclusion chromatography, hydrophobic interactionchromatography and/or by ion exchange chromatography, especially bycationic exchange chromatography.

The PEGylated IGF-I or IGF-I variant produced according to the inventionprovides improved stability in the circulation enabling a sustainedaccess to IGF-I receptors throughout the body with low applicationintervals.

The compounds of the present invention can be formulated according tomethods for the preparation of pharmaceutical compositions, whichmethods are known to the person skilled in the art. For the productionof such compositions, a PEGylated IGF-I or IGF-I variant according tothe invention is combined in a mixture with a pharmaceuticallyacceptable carrier, preferably by dialysis or diafiltration against anaqueous solution containing the desired ingredients of thepharmaceutical compositions. Such acceptable carriers are described, forexample, in Remington's Pharmaceutical Sciences, 18th edition, 1990,Mack Publishing Company, edited by Oslo et al. (e.g. pp. 1435-1712).Typical compositions contain an effective amount of the substanceaccording to the invention, for example from about 0.1 to about 100mg/ml, together with a suitable amount of a carrier. The compositionsmay be administered parenterally. The PEGylated IGF-I or IGF-I variantaccording to the invention is administered preferably viaintraperitoneal, subcutaneous, intravenous or intranasal application.

The pharmaceutical formulations according to the invention can beprepared according to known methods in the art. Usually, solutions ofPEGylated IGF-I or IGF-I variant are dialyzed or diafiltrated againstthe buffer intended to be used in the pharmaceutical composition and thedesired final protein concentration is adjusted by concentration ordilution.

The present invention relates also to a method for the treatment ofAlzheimer's disease comprising the administration of apharmaceutically-effective amount of lysine-PEGylated IGF-I orlysine-PEGylated IGF-I variant (as described above) to a patient in needof such treatment. In a preferred embodiment, the lysine-PEGylated IGF-Ior lysine-PEGylated IGF-I variant is administered in an amount of fromabout 0.1 to about 100 mg/ml.

The following examples and figures are provided to aid the understandingof the present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.Names of the amino acids are abbreviated using either the one lettercode (e.g. R) or the three letter code (e.g. Arg).

EXAMPLES Example 1 The Following Mutants (Comprising IGF-1-Variant RRK)with the Propeptides of the Amino Acid Sequence Met-X₁-His_(n)-X₂-Pro-Zwere Produced (PRPP Disclosed as SEQ ID NO: 29)

Mutant X₁ X₂ n Y Px3036_IAG none KAKRFKKH 6 PRPP K27R K65R K68(SEQ ID NO: 6) (SEQ ID NO: 2) Px3036_IAG_R none RARRFRRH 6 PRPPK27R K65R K68 (SEQ ID NO: 7) (SEQ ID NO: 2) Px3036_IAEE_F1 S NTEHNREH 6PRPP K27R K65R K68 (SEQ ID NO: 8) (SEQ ID NO: 3) Px3036_IAFX_F1 N IEGRH6 PRPP K27R K65R K68 (SEQ ID NO: 9) (SEQ ID NO: 4) Px3036_IAFX_F2 NTEFENIEH 6 PRPP K27R K65R K68 (SEQ ID NO: 10) (SEQ ID NO: 5)

Preparation of IGF-1-Variant (RRK Variant) monoPEGylated at ResiduePosition 68

The expression vector and the E. coli strain used are described in EP 0972 838. From an E. coli clone, expressing fusion protein px3036_IAG_RK27R K65R K68, px3036_IAEE_F1 K27R K65R K68, px3036_IAFX_F1 K27R K65RK68 or px3036_IAFX_F2 K27R K65R K68, grown on selective agar plate, oneinoculating loop was transferred to (100 ml) selective medium andcultivated for 13 h at 37° C. to an optical density (578 nm) of 2-4.This culture was stored on ice for the next 6 hours prior to theautomated inoculation of the main culture which was performed at 37° C.The expression of IGF-I mutant was initiated at an optical density (578nm) of 50 with the addition of 1.0 mM IPTG. The overall fermentationlasted up to 16 hours. The amount of protein was determineddensitometrically by comparing the volumetric intensity of the proteinband of the product with the band of an IGF standard on a SDS-PAGE gel.The culture broth was harvested by centrifugation.

To obtain purified inclusion body (IB) material, the harvested biomassout of standard fermentation was treated with the following procedure:biomass was resuspended with TrisMgSO₄ buffer pH7 and supplemented with0.3 g/100 g bio dry weight. Lysozyme and 5 U/1 g bio dry weightBenzonase were incubated for 20 min and homogenized. 30 U/1 g bio dryweight Benzonase was added and incubated for 60 min. at 37° C. 0.5 LBrij-buffer/liter was added and incubated for 30 min. at RT. Aftercentrifugation the pellet was resuspended in 300 ml Tris-EDTA-buffer/100g bio wet weight (purified IB wet weight), incubated for 30 min. at RTand centrifugated. 1 g IBs/liter were solubilized at room temperature in6.8 M guanidine-HCl, 0.1 M TrisHCl, 0.1 M DTT, pH 8.5 overnight. Theturbid solution was dialyzed at 4° C. against 6.8 M guanidine-HCl, 0.1 MTrisHCl, pH 8.0. After dialysis insoluble components were removed bycentrifugation. Folding was performed by 50-fold dilution of thepro-IGF-I solution into 0.8 M arginine, 0.1 M TrisHCl, 0.1 Mguanidine-HCl, 1 mM GSH, 1 mM GSSG, pH 8.5 at room temperature. Aftertwo hours the solution was supplemented with 2 M sodium chloride,filtered and applied at a flow rate of 10 ml/min to a HIC column (ButylSepharose 4 Fast Flow; GE, Amersham Biosciences), which had beenequilibrated at room temperature with buffer containing 2 M NaCl, 0.8 Marginine, 0.1 M TrisHCl, 0.1 M guanidine-HCl, pH 8.5. The column waswashed with equilibration buffer till baseline was achieved and theneluted with ten column volumes of a linear gradient starting withequilibration buffer and ending with buffer containing 0.1 M TrisHCl, 5%ethylene glycol, pH 8.5. Eluted fractions were analyzed by reversedphase high performance chromatography (rpHPLC). Fractions that containedprotein with correctly formed SS-bridges were pooled. The pool wasdialyzed at 4° C. against 50 mM sodium borate, pH 9.0. NHS activated 40kDa branched PEG (N-hydroxysuccinimide (NHS) ester of mPEG MW 20,000(mPEG2NHS, U.S. Pat. No. 5,932,462, Nektar Shearwater Polymers,Huntsville, Ala.) was solubilized in ice-cold 2 mM HCl and immediatelyadded to the dialyzed protein solution (molar PEG-reagent/protein ratio2:1). After 1 h and 2 h incubation on ice the same amount of acidicmPEG2-NHS solution was added to the protein/PEG reaction mix. After athird addition of the same amount of acidic mPEG2-NHS solution (overall6-fold molar excess of PEG reagent) the reaction was incubated on icefor an additional hour. The reaction was stopped by the addition ofsolid ammonium chloride and incubation for another 45 minutes and thenadjusted to pH 8.0 (see FIG. 1). The protein/PEG reaction mix wassupplemented with IgA1 protease from Neisseria gonorrhoea (type 2) (w/wratio 1:50) and incubated over night at room temperature (see FIG. 2).The reaction mix was diluted 1:2 with 50 mM acetic acid pH 4.5 and thenapplied to a cation IEC column (MacroCap SP support; GE, AmershamBiosciences, Uppsala, Sweden), which had been equilibrated with 50 mMacetic acid. The column was washed till baseline was reached and theneluted with 20 column volumes of a linear gradient starting with 50 mMacetic acid and ending with 50 mM acetic acid supplemented with 1 Msodium chloride. Eluted fractions were analyzed by SDS-PAGE. Fractionscontaining a single band with an estimated relative molecular size ofabout 60 kDa were pooled as IGF-I monoPEGylated at residue position 68(see FIG. 3). Identity of IGF-I monoPEGylated at residue position 68 wasverified by analytical size exclusion chromatography (SEC) with staticlight scattering detection, MS analysis of tryptic digests, MS analysisof Asp-N digests and analytical cation IEC. No other pegylationisovariant besides IGF-I monoPEGylated at residue position 68 could befound.

Example 2 The Following Mutants (Comprising IGF-1-Variant RKR) with thePropeptides of the Amino Acid Sequence Met-X₁-His_(n)-X₂-Pro-Z wereProduced (PRPP disclosed as SEQ ID NO: 29)

Mutant X₁ X₂ n Y Px3036_IAG none KAKRFKKH 6 PRPP K27R K65 K68R(SEQ ID NO: 6) (SEQ ID NO: 22) Px3036_IAG_R none RARRFRRH 6 PRPPK27R K65 K68R (SEQ ID NO: 7) (SEQ ID NO: 22) Px3036_IAEE_F1 S NTEHNREH 6PRPP K27R K65 K68R (SEQ ID NO: 8) (SEQ ID NO: 23) Px3036_IAFX_F1 N IEGRH6 PRPP K27R K65 K68R (SEQ ID NO: 9) (SEQ ID NO: 24) Px3036_IAFX_F2 NTEFENIEH 6 PRPP K27R K65 K68R (SEQ ID NO: 10) (SEQ ID NO: 25)

Preparation of IGF-1-Variant (RKR Variant) monoPEGylated at ResiduePosition 65

The expression vector and the E. coli strain for use are described in EP0 972 838. From an E. coli clone, expressing fusion protein px3036_IAG_RK27R K65 K68R, px3036_IAEE_F1 K27R K65 K68R, px3036_IAFX_F1 K27R K65K68R or px3036_IAFX_F2 K27R K65 K68R, grown on selective agar plate, oneinoculating loop is transferred to (100 ml) selective medium andcultivated for 13 h at 37° C. to an optical density (578 nm) of 2-4.This culture is stored on ice for the next 6 hours prior to theautomated inoculation of the main culture which is performed at 37° C.The expression of IGF-I mutant is initiated at an optical density (578nm) of 50 with the addition of 1.0 mM IPTG. The overall fermentationlasts up to 16 hours. The amount of protein is determineddensitometrically by comparing the volumetric intensity of the proteinband of the product with the band of an IGF standard on a SDS-PAGE gel.The culture broth is harvested by centrifugation.

To obtain purified inclusion body (IB) material, the harvested biomassout of standard fermentation is treated with the following procedure:biomass is resuspended with TrisMgSO4 buffer pH7, and supplemented with0.3 g/100 g bio dry weight Lysozyme and 5 U/1 g bio dry weight Benzonaseare incubated for 20 min and homogenized. 30 U/1 g bio dry weightBenzonase is added and incubated for 60 min. at 37° C. 0.5 LBrij-buffer/liter is added and incubated for 30 min. at RT. Aftercentrifugation the pellet is resuspended in 300 ml Tris-EDTA-buffer/100g bio wet weight (purified IB wet weight), incubated for 30 min. at RTand centrifugated. 1 g IBs/liter are solubilized at room temperature in6.8 M guanidine-HCl, 0.1 M TrisHCl, 0.1 M DTT, pH 8.5 overnight. Theturbid solution is dialyzed at 4° C. against 6.8 M guanidine-HCl, 0.1 MTrisHCl, pH 8.0. After dialysis insoluble components are removed bycentrifugation. Folding is performed by 50-fold dilution of thepro-IGF-I solution into 0.8 M arginine, 0.1 M TrisHCl, 0.1 Mguanidine-HCl, 1 mM GSH, 1 mM GSSG, pH 8.5 at room temperature. After 2to 48 hours, preferably after 2 to 24 hours the solution is supplementedwith 2 M sodium chloride, filtered and applied at a flow rate of 10ml/min to a HIC column (Butyl Sepharose 4 Fast Flow; GE, AmershamBiosciences), which is equilibrated at room temperature with buffercontaining 2 M NaCl, 0.8 M arginine, 0.1 M TrisHCl, 0.1 M guanidine-HCl,pH 8.5. The column is washed with equilibration buffer till baseline isachieved and then eluted with ten column volumes of a linear gradientstarting with equilibration buffer and ending with buffer containing 0.1M TrisHCl, 5% ethylene glycol, pH 8.5. Eluted fractions are analyzed byreversed phase high performance chromatography (rpHPLC). Fractions thatcontained protein with correctly formed disulfide-bridges are pooled.The pool is dialyzed at 4° C. against 50 mM sodium borate, pH 9.0. NHSactivated 40 kDa branched PEG (N-hydroxysuccinimide (NHS) ester of mPEGMW 20,000 (mPEG2NHS, U.S. Pat. No. 5,932,462, Nektar ShearwaterPolymers, Huntsville, Ala.) is solubilized in ice-cold 2 mM HCl andimmediately added to the dialyzed protein solution (molarPEG-reagent/protein ratio 2:1). After 1 h and 2 h incubation on ice thesame amount of acidic mPEG2-NHS solution is added to the protein/PEGreaction mix. After a third addition of the same amount of acidicmPEG2-NHS solution (overall 6-fold molar excess of PEG reagent) thereaction is incubated on ice for an additional hour. The reaction isstopped by the addition of solid ammonium chloride and incubation foranother 45 minutes and then adjusted to pH 8.0 (see FIG. 1). Theprotein/PEG reaction mix is supplemented with IgA1 protease fromNeisseria gonorrhoea (type 2) (w/w ratio 1:50) and incubated over nightat room temperature (see FIG. 2). The reaction mix is diluted 1:2 with50 mM acetic acid pH 4.5 and then applied to a cation IEC column(MacroCap SP support; GE, Amersham Biosciences, Uppsala, Sweden), whichis equilibrated with 50 mM acetic acid. The column is washed tillbaseline is reached and then eluted with 20 column volumes of a lineargradient starting with 50 mM acetic acid and ending with 50 mM aceticacid supplemented with 1 M sodium chloride. Eluted fractions areanalyzed by SDS-PAGE. Fractions containing a single band with anestimated relative molecular size of about 60 kDa are pooled as IGF-ImonoPEGylated at residue position 65. Identity of IGF-I monoPEGylated atresidue position 65 is verified by analytical size exclusionchromatography (SEC) with static light scattering detection, MS analysisof tryptic digests, MS analysis of Asp-N digests and analytical cationIEC.

Example 3 Reduction of Brain Soluble Abeta by IGF-I-variant RRKmonoPEGylated at Residue Position 68 In Vivo

For evaluation of potency of IGF-I variant RRK monoPEGylated at residueposition 68 (40 kD, PEG2) (PEG-RRK) on lowering soluble Abeta levels,9-10 months old B6.152H mice (doubletransgenic mice expressing human APPand PS2 mutants) with heavy amyloid plaque load were repeatedly treatedby twice-a-week s.c. injection of 5 μg/kg PEG-RRK.

Cortical APP, Abeta and Actin levels were detected after 14 days. TheAPP/actin ratio was not significantly changed by PEG-RRK (FIG. 4A)suggesting that PEG-RRK had no effect on transgene expression over 14days. In contrast, Abeta/Actin (FIG. 4B) and Abeta/APP (FIG. 4C) ratioswere significantly lowered by PEG-RRK. This indicates a positive effectof PEG-RRK on Abeta clearance independent on its production by thetransgene.

The invention claimed is:
 1. The method of preparing lysine-PEGylatedIGF-I or lysine-PEGylated IGF-I variant comprising (A) cultivating aprokaryotic host cell comprising an expression vector containing anucleic acid encoding a fusion protein comprising insulin-like growthfactor I (IGF-I) (SEQ ID NO: 1) or an IGF-I variant N-terminally linkedto the C-terminus of a propeptide, said C-terminus of said propeptidecomprising the amino acid sequence Y-Pro, wherein Y is selected from thegroup consisting of Pro; Pro-Ala; Pro-Gly; Pro-Thr; Ala-Pro; Gly-Pro;Thr-Pro; Arg-Pro; Pro-Arg-Pro; Ala-Pro-Arg-Pro (SEQ ID NO: 12); andPro-Ala-Pro-Arg-Pro (SEQ ID NO: 13) and said variant being a polypeptidewhich differs from SEQ ID NO: 1 in that one or two of the lysineresidues at positions 27, 65, or 68 thereof is independently substitutedby a polar amino acid selected from the group consisting of cysteine;aspartic acid; glutamic acid; histidine; asparagine; glutamine;arginine; serine; and threonine; and the remainder of the amino acidsequence of said variant is the same as that of SEQ ID NO: 1 and causingsaid cell to express said fusion protein; (B) recovering said fusionprotein; (C) lysine-PEGylating said fusion protein; (D) cleaving saidlysine-PEGylated fusion protein with IgA protease, and (E) recoveringsaid lysine-PEGylated IGF-I or lysine-PEGylated IGF-I variant producedin step (D).